Clostridial neurotoxins (NTs) have been extremely valuable for elucidating the mechanisms of neurotransmitter secretion from nerve cells since eight distinct NTs were found to act as specific proteases on only three neurosynaptic proteins: synaptobrevin, syntaxin and SNAP-25. However, a novel perspective has recently emerged from studies of NT action in non-neural cells. Once believed to be neural-specific, NTs introduced into the cytosol of non- neural cells by bypassing neural-specific cell-surface receptors are found to inhibit aspects of membrane trafficking. While surprising, these findings are understandable since a number of non-neural homologs of NT substrate proteins have been identified. The goals of this project are to develop a safe, efficient and general method for inducibly expressing Clostridial NT light chains in nonneural cells, and to employ this method to characterize the range of NT action on membrane trafficking in eukaryotic cells. The work proposed here will lead to the identification of specific membrane trafficking steps that are interrupted by NTs and to the characterization of NT substrates involved in these steps. Additional mutagenesis experiments are proposed to generate novel NT variants that have acquired a new substrate range. This work will also provide general tools for cell biological studies of commercial potential for investigating membrane trafficking in eukaryotic cells.